Genomic rearrangements involving the ETS transcription factor family genes are early driver events in prostate cancer (PCa). These rearrangements typically involve the fusion of androgen-regulated transcriptionally active genes with the ETS genes (ERG, ETV1, ETV4 and ETV5), resulting in fusion gene over-expression. The most prevalent ETS gene rearrangement, which is observed in >50% of PCa, involves the fusion of the androgen receptor (AR) target gene, TMPRSS2, with the ERG proto-oncogene, resulting in the formation of TMPRSS2- ERG gene fusion. Defining the mechanisms associated with the formation of recurrent genomic rearrangements will contribute towards our understanding of PCa etiology and will impact several aspects of clinical disease management ranging from prevention, early diagnosis and therapeutic targeting. The overall goal of this proposal is to apply innovative genomics approaches to define the origins of recurrent genomic rearrangements in PCa. On the basis of our work and the emerging body of scientific literature, we hypothesize that androgen signaling associated 3D genome organization and transcriptional regulation contribute towards topological stress in the genome. The enzyme DNA topoisomerase II beta (TOP2B) resolves topological stress by the formation of transient DNA double strand breaks (DSBs). We suggest that oxidative stress impairs the function of TOP2B, resulting in the formation of persistent DNA DSBs. Clustering of DNA DSBs in the genome increases the likelihood of their mis-repair and the formation of recurrent genomic rearrangements. In Specific Aim 1, we will define the AR-induced, transcription-associated chromatin interactions in PCa models. We will leverage this knowledge to pinpoint the mechanisms by which germ-line PCa predisposing risk SNPs and somatic structural variations alter the 3D genome architecture to drive transcriptional dysregulation in PCa. In Specific Aim 2, we will map the genomic regions associated with topological stress by applying our new strategy to identify TOP2B occupancy. We will also test the role of oxidative stress and TDP2, a 5-prime tyrosyl-DNA phosphodiesterase in the formation of TOP2B mediated DNA breaks. Furthermore, we will test the hypothesis that TOP2B mediated DNA DSBs are non-random and occur at distinct genomic regions in the various cell types of the prostate?dictated in part by transcription-associated 3D genome architecture. Therefore, in Specific Aim 3, we will unravel the relationship between prostate epithelial differentiation status and the formation of TMPRSS2-ERG gene fusions. We anticipate that successful completion of these aims will enhance our understanding of the origins of recurrent genomic rearrangements in PCa. We believe that our discoveries in PCa models will have broad implications for many other types of cancers. Importantly, these studies will serve as the rationale for future clinical trials aimed towards the prevention and treatment of PCa.